Device containing cytophilic islands that adhere cells separated by cytophobic regions

ABSTRACT

The invention provides a device for adhering cells in a specific and predetermined position, and associated methods. The device includes a plate defining a surface and a plurality of cytophilic islands that adhere cells, isolated by cytophobic regions to which cells do not adhere, contiguous with the cytophilic islands. The islands or the regions or both may be formed of a self-assembled monolayer (SAM).

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/373,334, filed Aug. 12, 1999, now U.S. Pat. No. 6,368,838 which is acontinuation of U.S. patent application Ser. No. 08/951,886, filed Oct.16, 1997, now U.S. Pat. No. 5,976,826, which is a continuation of U.S.patent application Ser. No. 08/659,537, filed Jun. 7, 1996, now U.S.Pat. No. 5,776,748, which is a continuation of U.S. patent applicationSer. No. 08/131,838 filed Oct. 4, 1993, now abandoned.

This invention was made with government support NSF Grant NumberEEC-880-3014, NIH Grant Number GM30367, ONR Grant NumberN00014-86-K-0756, and ACS Grant Number CD-493. The government hascertain rights to the invention.

FIELD OF THE INVENTION

The present invention relates generally to derivatization and patterningof surfaces, more particularly to the formation on surfaces of patternsof self-assembled molecular monolayers with differing bioadhesiveproperties using a microstamp, novel articles produced thereby, and usestherefor.

BACKGROUND OF THE INVENTION

In the field of microelectronic devices and sensors, the development ofdevices that are small relative to the state of the art, convenientlyand relatively inexpensively reproduced, and produced with a relativelylow failure rate has long been important. In the fields of cellular anddevelopmental, and molecular biology, microbiology, biomedical devices,and biotechnology, there is now a growing need for devices of similarscale with features as small as or smaller than individual cells.

In the electronics industries, such devices have been produced by avariety of methods. A well-known method of production of such devices isphotolithography. According to this technique, a thin film ofconducting, insulating, or semiconducting material is deposited on asubstrate and a negative or positive resist (photoresist) is coated ontothe exposed surface of the material. The resist is then irradiated in apredetermined pattern, and irradiated (positive resist) ornon-irradiated (negative resist) portions of the resist are washed fromthe surface to produce a predetermined pattern of resist on the surface.Alternatively, micromachining has been employed to mechanically removesmall areas from a surface to form a pattern.

While the above-described irradiative lithographic methods may beadvantageous in many circumstances, all require relatively sophisticatedand expensive apparatus to reproduce a particular material pattern on aplurality of substrates, and are relatively time-consuming.Additionally, no method of patterning other than on a flat substrate iscommonly available according to the methods.

These techniques have recently been employed in the biological sciencesto create patterned surfaces on which cells may be adhered and grown.For example, the orientation, spreading, and shape of several cell typeshave been shown to be affected by topography. Thus cells have been grownon grooved surfaces which have been created by micromachining surfacesor by using photolithography to etch away parts of surfaces. (See, forexample, D. M. Brunette, Exp. Cell Res., 167:203-217, 1986; T. Inoue, etal., J. Biomedical Materials Res., 21:107-126, 1987; B. Chehroudi, etal., J. Biomedical Materials Res., 22:459-473, 1988; G. A. Dunn and A.F. Brown, J. Cell Sci., 83:313-340, 1986; A. Wood, J. Cell Sci.,90:667-681, 1988; B. Chehroudi, et al., J. Biomedical Materials Res.,24-1203-1219, 1990; P. Clark, et al. Development, 99:439-448, 1987.

A need exists in the art for a convenient, inexpensive, and reproduciblemethod of plating or etching a surface according to a predeterminedpattern. The method would ideally find use on planar or nonplanarsurfaces, and would result in patterns having features in the submicrondomain. Additionally, the method would ideally provide for convenientreproduction of existing patterns.

The study of self-assembled monolayers (SAMs) is an area of significantscientific research. Such monolayers are typically formed of moleculeseach having a functional group that selectively attaches to a particularsurface, the remainder of each molecule interacting with neighboringmolecules in the monolayer to form a relatively ordered array. Such SAMshave been formed on a variety of substrates including metals, silicondioxide, gallium arsenide, and others. SAMs have been applied tosurfaces in predetermined patterns in a variety of ways including simpleflooding of a surface and more sophisticated methods such as irradiativepatterning.

Monolayers may be produced with varying characteristics and with variousfunctional groups at the free end of the molecules which form the SAM.Thus, SAMs may be formed which are generally hydrophobic or hydrophilic,generally cytophobic or cytophilic, or generally biophobic or biophilic.Additionally, SAMs with very specific binding affinities can beproduced. This allows for the production of patterned SAMs which willadhere cells, proteins, or other biological materials in specific andpredetermined patterns.

Accordingly, a general purpose of the present invention is to provide amethod of conveniently and reproducibly producing a variety of SAMpatterns on planar as well as nonplanar surfaces, the patterns havingresolution in the submicron domain and being capable of adhering cells,proteins, or other biological materials in specific and predeterminedpatterns. Another purpose of the invention is to provide a method offorming a template from an existing pattern having micron orsubmicron-domain features, the template conveniently reproducing thepre-existing pattern.

SUMMARY OF THE INVENTION

The invention provides novel devices useful for adhering cells inspecific and predetermined positions. Such devices are useful in a widearray of cellular biology applications, including cell culturing,recombinant protein production, cytometry, toxicology, cell screening,microinjection, immobilization of cells, influencing the state ofdifferentiation of a cell including promoting differentiation, arrestingdifferentiation or causing dedifferentiation. The devices of theinvention also can be used to promote ordered cell-cell contact or tobring cells close to one another, but prevent such contact. The devicesof the invention also are useful in the creation of artificial tissuesfor research or in vivo purposes and in connection with creatingartificial organs such as artificial liver devices. The devices also areuseful in connection with generating surfaces for prosthetic orimplantable devices.

According to one aspect of the invention, a plate defining a surfacewith a cytophilic island is provided. The cytophilic island includes aself-assembled monolayer. In one preferred embodiment, the deviceincludes a plurality of such islands. These islands can be isolated by acytophobic region including a self-assembled monolayer, which can becontiguous with the cytophilic island.

According to another aspect of the invention, the plate has a surfacewith a cytophilic island, wherein the cytophilic island is isolated by acytophobic region including a self-assembled monolayer, which can becontiguous with the cytophilic island. Again, preferred devices includea plurality of such islands.

Islands of the foregoing type can take on virtually any shape whenmanufactured according to the methods of the invention, includingelongated shapes. They also can be adapted to bind only selected celltypes. Preferred islands are between 1 and 2,500 square microns,preferably between 1 and 500 square microns. In some applications, theislands can have an area of as little as between 1 and 100 squaremicrons. Also according to the invention, the islands may have a lateraldimension of between 0.2 and 10 microns. According to another aspect ofthe invention, the plate has a surface and a pair of cytophilic regions.Each of the cytophilic regions includes a self-assembled monolayer. Oneof the pair has its self-assembled monolayer modified so as to bind afirst cell type but not a second cell type, and the other of the pairhas its self-assembled monolayer modified so as to bind the second celltype but not the first cell type. Such cytophilic regions can bepositioned to promote or prevent cell-cell contact. They in particularcan be adapted to prevent cell-cell contact, but be close enough so theypermit cell-cell communication via secreted molecules such as cytokines.

The invention in its broadest aspects including methods and productsuseful in manufacturing the devices will be described in greater detailbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a schematic illustration of the stamping process of thepresent invention and one potential embodiment, including photocopies ofscanning electron microscopy (SEM) figures of an exemplary stampingsurface, a SAM pattern formed according to the inventive process, andcells adhered to specific and predetermined sites on one of the platesof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for producing patterned surfacesfor plating cells, proteins, or other biological materials in a specificand predetermined pattern. In particular, it provides a method ofproducing plates with patterned regions of material capable of bindingcells, proteins or other biological materials, interspersed with regionsof material incapable of binding such biological samples. Significantly,the present invention provides for the production of patterned plates inwhich the dimensions of the features or details of the patterns may beas small as 0.2-1 μm.

The invention derives from a general new method of creating patternedsurfaces applicable in a variety of fields. The method is simple andprovides for relatively inexpensive production of many copies of thepatterned surface. Applications of the method relating to the productionof microelectronic devices are described in U.S. patent application Ser.No. 08/131,841 filed by Kumar and Whitesides on Oct. 4, 1993 andentitled “Formation of Microstamped Patterns on Surfaces and DerivativeArticles”, the disclosure of which is incorporated herein by reference.

The patterns of the present invention are formed by self-assembledmonolayers (SAMs) of organic molecules which spontaneously chemisorb tothe surface of a plate. The SAM patterns are applied to the plate usinga stamp in a “printing” process in which the “ink” consists of asolution including a compound capable of chemisorbing to form a SAM. Theink is applied to the surface of a plate using the stamp and deposits aSAM on the plate in a pattern determined by the pattern on the stamp.The plate may be stamped repeatedly with the same or different stamps invarious orientations and with the same or different SAM-formingsolutions. In addition, after stamping, the portions of the plate whichremain bare or uncovered by a SAM may be derivatized. Suchderivatization may conveniently include exposure to another solutionincluding a SAM-forming compound. The SAM-forming or derivatizingsolutions are chosen such that the regions of the finished plate definedby the patterns differ from each other in their ability to bindbiological materials. Thus, for example, a grid pattern may be createdin which the square regions of the grid are cytophilic and bind cellsbut the linear regions of the grid are cytophobic and no cells bind tothese regions.

A simple illustration of the general process underlying the presentinvention is presented in FIG. 1. FIG. 1(a) shows an polymeric material20 cast onto a mold with raised features 44 defining a pattern. FIG.1(b) shows the stamp 20 with stamping surface 26 after curing andseparation from the mold. FIG. 1(c) shows the stamp after inking with anink 28 including a SAM-forming compound. FIG. 1(d) shows a platecomprising a substrate 34 and a thin coating of surface material 32after stamping. The SAM forming compound of the ink has chemisorbed tothe material surface 32 to form a SAM 35 with surface regions 36 in apattern corresponding to the stamping surface 26 of the stamp 20. FIG.1(e) shows the plate after exposure to a second or filling solutionincluding a SAM-forming compound. The second solution has filled thebare regions 37 of the surface material 32 with a second or filling SAM38. FIG. 1(f) shows an electron micrograph of the surface of a typicalplate formed in a grid pattern as shown according to the stepsrepresented in FIG. 1(a) through FIG. 1(e). FIG. 1(g) shows an platewith a patterned SAM on which a material 39 has selectively bound to thesurface regions 36 of the first SAM 35. FIG. 1(h) is an electronmicrograph of the grid patterned SAM of FIG. 1(f) after exposure to aprotein which bound only to the square islands. FIG. 1(i) shows a SAM onwhich a material 39 has bound to the surface 36 of a SAM and cells 40have bound to the material 39. FIG. 1(j) shows an electron micrograph ofa SAM with cells bound at specific and predetermined positions.

The mold used to form the stamp may be a commercially available itemsuch as a transmission electron microscopy grid or any other corrugatedmaterial possessing a pattern which is desired to be reproduced on thestamp. Alternatively, the mold may be especially prepared by any of avariety of methods known in the art. According to one, the mold surfaceis micromachined from a material such as metal. According to another,the mold surface is formed lithographically by providing a substrate,depositing a film of material onto the substrate, coating an exposedsurface of the material with resist, irradiating the resist according toa predetermined pattern, removing irradiated portions of the resist fromthe material surface, contacting the material surface with a reactantselected to react chemically therewith and selected to be chemicallyinert with respect to the resist such that portions of the materialaccording to the predetermined pattern are degraded, removing thedegraded portions, and removing the resist to uncover portions of thematerial formed according to the predetermined pattern to form the moldsurface. Negative or positive resist may be used, and the procedureadjusted accordingly.

According to another method of forming a mold surface, a substrate maybe provided, and coated with resist. Then portions of the resist may beirradiated according to a particular predetermined pattern. Irradiatedportions of the resist may then be removed from the substrate to exposeportions of the substrate surface according to the predeterminedpattern, and the substrate may be contacted with a plating reagent suchthat exposed portions according to the predetermined pattern are plated.Then, the resist may be removed to uncover portions of the exposedsubstrate according to the predetermined pattern bordered by platedportions of the substrate to form the mold surface.

As noted above, however, any corrugated material may be used as a moldto form the stamps of the present invention.

The stamp is produced by casting a polymeric material onto a mold havingthe desired pattern. The particular material chosen for formation of thestamp is not critical to the present invention, but should be chosen soas to satisfy certain physical characteristics. The stamp isadvantageously chosen to be elastic, such that the stamping surface mayvery closely conform to minute irregularities in the surface material ofthe plate to be stamped and to completely transfer the ink thereto, andso as to be amenable to transferring SAMs to nonplanar surfaces. Thestamp should not, however, be so elastic as to greatly deform in shapeduring stamping as this will cause a blurring of the desired pattern.The stamp should also be formed such that the stamping surface comprisesan absorbent material selected to absorb SAM-forming solutions. Thematerial may also be swellable. Such swelling and absorbingcharacteristics serve the important function of providing gooddefinition of an isolated SAM on the surface material of the plate.

For example, if a dimensional feature of the stamping surface includes asubstantially square-shaped feature, the stamping surface shouldtransfer a SAM-forming compound to the surface material of the plate soas to form SAMs mirroring the substantially square features of thestamping surface, without blurring. Such blurring results from selectionof a stamp which does not absorb the ink. When such a stamp is employed,the ink resides as a liquid on the stamping surface, rather thanpartially or fully within the stamping surface, and when the stampingsurface contacts the surface material of a plate, the ink is dispersedfrom under the stamping surface. According to the stamp of the presentinvention, however, the ink is dry both on and within the stampingsurface. The ink may be dried by simple exposure to air or, if desired,an air current may be provided by a blower or jet. Thus, the ink isabsorbed into the stamping surface, dries, and when the stamping surfacecontacts the surface material of a plate, the ink is not dispersed, butbinds to the plate, and the removal of the stamping surface from thesurface material of the plate results in well-defined SAM features.

Additionally, the stamp should be fabricated such that the stampingsurface is free of any leachable materials such as plasticizers thatwould interfere with or contaminate the ink. For example, if additivesare included in the material used to fabricate the stamp, such additivesshould be bound to the internal structure of the stamp. For example, ifthe stamp is fabricated from a polymeric material, any additives shouldbe bound to the polymer backbone thereof.

Material selected for use in fabrication of the stamp is advantageouslyselected so as not to undergo substantial shape changes when the stampis formed. For example, when a hardenable fluid is brought into contactwith the mold and is hardened, little or no shape change should takeplace upon such hardening. Thus, any shape changes of features of thestamping surface should be within tolerable limits of precision forformation of SAM features transferred to the surface material of theplate by the stamping surface. If any shape changes do occur uponhardening of the material selected for fabrication of the stamp, it maybe desirable that such changes involve miniaturization of the stampingsurface features.

According to a preferred embodiment, the stamp is formed from apolymeric material. Polymeric materials suitable for use in thefabrication of the stamp may have linear or branched backbones, and maybe crosslinked or non-crosslinked, depending upon the particular polymerand the degree of formability desired of the stamp. A variety ofelastomeric polymeric materials are suitable for such fabrication,especially polymers of the general classes of silicone polymers andepoxy polymers. Epoxy polymers are characterized by the presence of athree-member cyclic ether group commonly referred to as an epoxy group,1,2-epoxide, or oxirane. For example, diglycidyl ethers of bisphenol Amay be used, in addition to compounds based on aromatic amine, triazine,and cycloaliphatic backbones. Another example includes the well-knownNovolac polymers.

Examples of silicone elastomers suitable for use as the stamp includethose formed from precursors including the chlorosilanes such asmethylchlorosilanes, ethylchlorosilanes, and phenylchlorosilanes, andthe like. A particularly preferred silicone elastomer ispolydimethylsiloxane (PDMS). This material has produced better stampsthan polyethylene or polystyrene, which were insufficiently elastic, andpolybutadine, which was too elastic.

In addition to the above-described methods for forming the stamp, aphotolytic method may be employed in the present invention. For example,a mask may be positioned between a surface and a source of irradiation,and the surface irradiated through the mask for a predetermined periodof time. Portions of the surface may be degraded by such irradiation,forming indentations in the surface upon removal of such degradedportions, and thereby defining a stamping surface. According to thismethod, a variety of patterns may be very conveniently formed in a stampaccording to a variety of available masks. In addition, the photolyticmethod may be used in combination with the above-described methodsinvolving hardening a hardenable fluid on a mold surface. For example, ahardenable fluid may be contacted with a mold surface and allowed toharden to form a stamp having a first predetermined stamping surface,and then the first predetermined stamping surface is irradiated througha mask to create additional features in the stamping surface.

The stamp includes a stamping surface having a variety of featuresdefined by the border between the stamping surface and indentations inthe surface. According to the invention, the stamping surface mayinclude features having a variety of lateral dimensions, including verylarge lateral dimensions for transferring a large SAM to a surface.According to some embodiments of the invention, however, it is highlyadvantageous to fabricate stamping surfaces so as to have at least onefeature with a lateral dimension of less than about 100 microns so as tobe able to produce SAMs capable of binding single cells or narrow bandsof biological materials such as proteins. According to otherembodiments, the stamping surface may include at least one feature witha lateral dimension of less than about 50 microns, less than about 10microns, less than about 5 microns, less than about 1 micron, or lessthan about 0.25 microns.

The stamp is inked with a solution capable of forming a SAM bychemisorption to a surface. The inking may, for example, be accomplishedby (1) contacting the stamp with a piece of lint-free paper moistenedwith the ink, (2) pouring the ink directly onto the stamp or (3)applying the ink to the stamp with a cotton swab. The ink is thenallowed to dry on the stamp or is blown dry so that no ink in liquidform, which may cause blurring, remains on the stamp. The SAM-formingcompound may be very rapidly transferred to the stamping surface. Forexample, contacting the stamping surface with the compound for a periodof time of approximately 2 seconds is generally adequate to effectsufficient transfer, or contact may be maintained for substantiallylonger periods of time. The SAM-forming compound may be dissolved in asolvent for such transfer, and this is often advantageous in the presentinvention. Any organic solvent within which the compound dissolves maybe employed but, preferably, one is chosen which aids in the absorptionof the SAM-forming compound by the stamping surface. Thus, for example,ethanol, THF, acetone, diethyl ether, toluene, isooctane and the likemay be employed. For use with a PDMS stamp, ethanol is particularlypreferred, and toluene and isooctane and not preferred as they are notwell absorbed. The concentration of the SAM-forming compound in the inksolution may be as low as 1 μM. A concentration of 1-10 mM is preferredand concentrations above 100 mM are not recommended.

The plate is then contacted with the stamp such that the inked stampingsurface bearing the pattern contacts the surface material of the plate.This may be accomplished by hand with the application of slight fingerpressure or by a mechanical device. The stamp and plate need not be heldin contact for an extended period; contact times between 1 second and 1hour result in apparently identical patterns for hexadecanethiol (1-10mM in ethanol) ink applied to a plate with a gold surface. Duringcontact, the SAM-forming compound of the ink reacts with the surface ofthe plate such that, when the stamp is gently removed, a SAM ischemisorbed to the plate in a pattern corresponding to the stamp.

As used herein, a “plate” means any object with a surface capable ofreacting with a solution including a SAM-forming compound such that aSAM is formed on the surface. The plate may be flat as in a tissueculture plate or glass slide. The plate may also, however, becorrugated, rugose, concave, convex or any combination of these. Theplate may also be a prosthetic or implantable device on which it isdesired to form a SAM or adhere patterns of cells, proteins, or otherbiological materials. The word “plate” is used only for expositorybrevity and is not to be construed as limiting the scope or claims ofthe present invention to planar surfaces.

A variety of compounds may be used in solution as the ink and a varietyof materials may provide the surface material onto which the ink isstamped and the SAM is formed. In general, the choice of ink will dependon the surface material to be stamped. In general, the surface materialand SAM-forming compound are selected such that the SAM-forming compoundterminates at a first end in a functional group that binds or chemisorbsto the surface of the surface material. As used herein, the terminology“end” of a compound is meant to include both the physical terminus of amolecule as well as any portion of a molecule available for forming abond with the surface in a way that the compound can form a SAM. Thecompound may comprise a molecule having first and second terminal ends,separated by a spacer portion, the first terminal end comprising a firstfunctional group selected to bond to the surface material of the plate,and the second terminal end optionally including a second functionalgroup selected to provide a SAM on the material surface having adesirable exposed functionality. The spacer portion of the molecule maybe selected to provide a particular thickness of the resultant SAM, aswell as to facilitate SAM formation. Although SAMs of the presentinvention may vary in thickness, as described below, SAMs having athickness of less than about 50 Angstroms are generally preferred, morepreferably those having a thickness of less than about 30 Angstroms andmore preferably those having a thickness of less than about 15Angstroms. These dimensions are generally dictated by the selection ofthe compound and in particular the spacer portion thereof.

A wide variety of surface materials and SAM-forming compounds aresuitable for use in the present invention. A non-limiting exemplary listof combinations of surface materials and functional groups which willbind to those surface materials follows. Although the following listcategorizes certain preferred materials with certain preferredfunctional groups which firmly bind thereto, many of the followingfunctional groups would be suitable for use with exemplary materialswith which they are not categorized, and any and all such combinationsare within the scope of the present invention. Preferred materials foruse as the surface material include metals such as gold, silver, copper,cadmium, zinc, palladium, platinum, mercury, lead, iron, chromium,manganese, tungsten, and any alloys of the above when employed withsulfur-containing functional groups such as thiols, sulfides,disulfides, and the like; doped or undoped silicon employed with silanesand chlorosilanes; metal oxides such as silica, alumina, quartz, glass,and the like employed with carboxylic acids; platinum and palladiumemployed with nitrites and isonitriles; and copper employed withhydroxamic acids. Additional suitable functional groups include acidchlorides, anhydrides, sulfonyl groups, phosphoryl groups, hydroxylgroups and amino acid groups. Additional surface materials includegermanium, gallium, arsenic, and gallium arsenide. Additionally, epoxycompounds, polysulfone compounds, plastics and other polymers may finduse as the surface material in the present invention. Polymers used toform bioerodable articles, including but not limited to polyanhydrides,and polylactic and polyglycolic acids, are also suitable. Additionalmaterials and functional groups suitable for use in the presentinvention can be found in U.S. Pat. No. 5,079,600, issued Jan. 7, 1992,and incorporated herein by reference.

According to a particularly preferred embodiment, a combination of goldas the surface material and a SAM-forming compound having at least onesulfur-containing functional group such as a thiol, sulfide, ordisulfide is selected.

The SAM-forming compound may terminate in a second end, opposite to theend bearing the functional group selected to bind to the surfacematerial, with any of a variety of functionalities. That is, thecompound may include a functionality that, when the compound forms a SAMon the surface material, is exposed. Such a functionality may beselected to create a SAM that is hydrophobic, hydrophilic, thatselectively binds various biological or other chemical species, or thelike. For example, ionic, nonionic, polar, nonpolar, halogenated, alkyl,aryl or other functionalities may exist at the exposed portion of thecompound. A non-limiting, exemplary list of such functional groupsincludes those described above with respect to the functional group forattachment to the surface material in addition to: —OH, —CONH—,—CONHCO—, —NH₂, —NH—, —COOH, —COOR, —CSNH—, —NO₂ ⁻, —SO₂ ⁻, —RCOR—,—RCSR—, —RSR, —ROR—, —PO₄ ⁻³, —OSO₃ ⁻², —SO₃ ⁻, —NH_(x)R_(4−x) ⁺, —COO⁻,—SOO⁻, —RSOR—, —CONR₂, —(OCH₂CH₂)_(n)OH (where n=1-20, preferably 1-8),—CH₃, —PO₃H⁻, -2-imidazole, —N(CH₃)₂, —NR₂, —PO₃H₂, —CN, —(CF₂)_(n)CF₃(where n=1-20, preferably 1-8), olefins, and the like. In the abovelist, R is hydrogen or an organic group such as a hydrocarbon orfluorinated hydrocarbon. As used herein, the term “hydrocarbon” includesalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, and thelike. The hydrocarbon group may, for example, comprise methyl, propenyl,ethynyl, cyclohexyl, phenyl, tolyl, and benzyl groups. The term“fluorinated hydrocarbon” is meant to refer to fluorinated derivativesof the above-described hydrocarbon groups.

In addition, the functional group may be chosen from a wide variety ofcompounds or fragments thereof which will render the SAM generally orspecifically “biophilic” as those terms are defined below. Generallybiophilic functional groups are those that would generally promote thebinding, adherence, or adsorption of biological materials such as, forexample, intact cells, fractionated cells, cellular organelles,proteins, lipids, polysaccharides, simple carbohydrates, complexcarbohydrates, and/or nucleic acids. Generally biophilic functionalgroups include hydrophobic groups or alkyl groups with charged moietiessuch as —COO⁻, —PO₃H^(− or) 2-imidazolo groups, and compounds orfragments of compounds such as extracellular matrix proteins,fibronectin, collagen, laminin, serum albumin, polygalactose, sialicacid, and various lectin binding sugars. Specifically biophilicfunctional groups are those that selectively or preferentially bind,adhere or adsorb a specific type or types of biological material so as,for example, to identify or isolate the specific material from a mixtureof materials. Specific biophilic materials include antibodies orfragments of antibodies and their antigens, cell surface receptors andtheir ligands, nucleic acid sequences and many others that are known tothose of ordinary skill in the art. The choice of an appropriatebiophilic functional group depends on considerations of the biologicalmaterial sought to be bound, the affinity of the binding required,availability, facility of ease, effect on the ability of the SAM-formingcompound to effectively form a SAM, and cost. Such a choice is withinthe knowledge, ability and discretion of one of ordinary skill in theart.

Alternatively, the functional group may be chosen from a wide variety ofcompounds or fragments thereof which will render the SAM “biophobic” asthat term is defined below. Biophobic SAMs are those with a generallylow affinity for binding, adhering, or adsorbing biological materialssuch as, for example, intact cells, fractionated cells, cellularorganelles, proteins, lipids, polysaccharides, simple carbohydrates,complex carbohydrates, and/or nucleic acids. Biophobic functional groupsinclude polar but uncharged groups including unsaturated hydrocarbons. Aparticularly preferred biophobic functional group is polyethylene glycol(PEG).

The central portion of the molecules comprising the SAM-forming compoundgenerally includes a spacer functionality connecting the functionalgroup selected to bind the to surface material and the exposedfunctionality. Alternately, the spacer may essentially comprise theexposed functionality, if no particular functional group is selectedother than the spacer. Any spacer that does not disrupt SAM packing andthat allows the SAM layer to be somewhat impermeable to various reagentssuch as etching reagents, as described below, in addition to organic oraqueous environments, is suitable. The spacer may be polar; non-polar;halogenated or, in particular, fluorinated; positively charged;negatively charged; or uncharged. For example, a saturated orunsaturated, linear or branched alkyl, aryl, or other hydrocarbon spacermay be used.

A variety of lengths of the SAM-forming compound may be employed in thepresent invention. If two or more different SAM-forming compounds areused in one stamping step, for example if two or more differentSAM-forming compounds are used in the ink, it is often advantageous thatthese species have similar lengths. However, when a two or more stepprocess is used in which a first SAM is provided on a surface and atleast a second SAM is provided on the surface, the various SAMs beingcontinuous or noncontinuous, it may be advantageous in somecircumstances to select molecular species for formation of the variousSAMs that have different lengths. For example, if the SAM formed bystamping has a first molecular length and the SAM subsequentlyderivatized to the surface has a second molecular length greater thanthat of the stamped SAM, a continuous SAM having a plurality of “wells”results. These wells are the result of the stamped SAM being surroundedby the second SAM having a longer chain length. Such wells may beadvantageously fabricated according to certain embodiments, for example,when it is desirable to add greater lateral stability to particularbiological materials, such as cells, which have been captured in thewells. Such wells may also form the basis for reaction vessels.

Additionally, SAMs formed on the surface material may be modified aftersuch formation for a variety of purposes. For example, a SAM-formingcompound may be deposited on the surface material in a SAM, the compoundhaving an exposed functionality including a protecting group which maybe removed to effect further modification of the SAM. For example, aphotoremovable protecting group may be used, the group beingadvantageously selected such that it may be removed without disturbanceof the SAM of which it is a part. For example, a protective group may beselected from a wide variety of positive light-reactive groupspreferably including nitroaromatic compounds such as o-nitrobenzylderivatives or benzylsulfonyl. Photoremovable protective groups aredescribed in, for example, U.S. Pat. No. 5,143,854, issued Sep. 1, 1992,and incorporated herein by reference, as well as an article byPatchomik, JACS, 92, 6333 (1970) and Amit et al., JOC, 39, 192, (1974),both of which are incorporated herein by reference. Alternately, areactive group may be provided on an exposed portion of a SAM that maybe activated or deactivated by electron beam lithography, x-raylithography, or any other radiation. Such protections and deprotectionsmay aid in chemical or physical modification of an existingsurface-bound SAM, for example in lengthening existing molecular speciesforming the SAM. Such modification is described in U.S. Pat. No.5,143,857, referenced above.

The preferred surface portions are cytophilic, that is, adapted topromote cell attachment. Molecular entities creating cytophilic surfacesare well known to these of ordinary skill in the art and includeantigens, antibodies, cell adhesion molecules, extracellular matrixmolecules such as laminin, fibronectin, synthetic peptides,carbohydrates and the like.

The surface material of the plate may comprise the entire plate ontowhich the patterned SAMs of the present invention are chemisorbed, ormay be a thin film deposited upon a substrate. Where a separatesubstrate is employed, it may comprise any biological, non-biological,organic, or inorganic material, or a combination of any of theseexisting as particles, strands, precipitates, gels, sheets, tubing,spheres, containers, capillaries, pads, slices, films, slides, etc.Generally, the substrate of the present invention is substantiallyplanar, although it need not be according to certain embodiments. Thesubstrate may be formed of a conductive, non-conductive, semiconductingmaterial, or the like, and may comprise glass, silica, alumina, plasticor other organic polymers including acrylonitrile-butadine-styrenecopolymers, polysulfone, metals, as well as bioerodable polymersincluding polyanhydrides or polylactic or polyglycolic acids, or any ofthe above materials described with respect to the surface material ofthe present invention. The substrate may additionally include a bondinglayer, for example a thin titanium film, to promote adhesion between thesurface material and the substrate.

The surface material is generally of a thickness on the order of 500microns, but may be substantially thicker or may be substantiallythinner. For example, when a substrate as a base material is employed,the surface material may have a thickness of less than about 100nanometers, less than about 10 nanometers, or even less than about 6nanometers. When a very thin film of surface material is employed, and atransparent substrate supports the surface material, a transparent basesupport for a SAM results, and this may be advantageous in standardlight or electron microscopic or spectrophotometric detection oranalysis of any biological material interacting with a SAM on thesurface material.

Now that a detailed description of the process for producing patternedplates for plating cells, proteins, and other biological materials hasbeen provided, a variety of particular preferred embodiments relating toparticular plate patterns and their uses are disclosed below. Theseembodiments are intended to be illustrative and are not intended tolimit the uses to which the plates of the present invention may beapplied.

In a first series of embodiments, a patterned plate is produced withbiophilic “islands.” By “islands”, as used herein, is meant regions ofbiophilic SAM surrounded by biophobic SAM. Thus, islands on a plate areregions to which cells, proteins or other biological materials may beexpected to adhere or bind. Islands may be of any size or shape,including rectilinear, circular, ovoid and arbitrary shapes. In somepreferred embodiments, islands are of such area and shape so as topermit binding of only a single cell of a given type or types. In otherembodiments, as described below, islands may be shaped or sized so as tocreate desired patterns of a multiplicity of adhered cells in contactwith one another on a given island but separated from and not in contactwith cells on a different island. Irrespective of the shape of theislands, a pattern consisting of an array of islands is referred toherein as a grid pattern.

As noted above, the same plate may be stamped several times. In oneembodiment, a grid of islands is produced by employing a stampconsisting of a pattern of parallel lines. This stamp may be contactedwith the plate a first time in a first orientation and then, afterreinking the stamp if necessary, either the stamp or the plate isrotated through some angle and contacted a second time in a secondorientation. If the angle of rotation is 90°, a square grid pattern maybe produced.

After the desired SAM pattern has been formed on the plate, the portionof the plate's surface which is bare or not covered by the stamped SAMmay be derivatized by exposing it to a second or “filling” solution withcharacteristics differing from the first solution which was used as theink. This exposure may be accomplished by dipping the plate in a bath ofsolution, by pouring the solution onto the plate or by any otherconvenient method which does not disrupt the patterned SAM. The secondsolution may form a SAM over the surface of the plate which is notalready covered by the patterned SAM of the ink. That is, the second offilling solution may contain a second or “filling” SAM-forming compoundwhich will form a second or “filling” SAM on the bare portions of theplate's surface. The result is a plate completely covered bycomplementary patterns of two or more SAMs of differing properties.

In this embodiment of forming a grid pattern, the ink used in thestamping can form a biophobic SAM or it can be modified in situ afterSAM formation to form a biophobic SAM by reacting the functional groupsat the free ends of the SAM with a compound that will make it biophobic.The filling solution, which derivatizes the areas of the platecorresponding to the islands, can form a biophilic SAM or it can bemodified in situ after SAM formation to form a biophilic SAM by reactingthe functional groups at the free ends of the SAM with a compound thatwill make it biophilic.

In an alternative embodiment, the raised stamping surface of the stampmay correspond to the islands and the ink will form a SAM which isbiophilic or may be made biophilic by modifying the functional groups atthe free ends of the SAM. In this embodiment, the filling solution whichderivatizes the bare regions of the surface material can form abiophobic filling SAM or may be modified so as to become biophobic. Inthis embodiment, round, oval or arbitrarily shaped, in addition tosquare or rectangular islands may be produced.

It is not necessary in all embodiments to derivatize any bare portionsof the surface remaining after forming patterned SAMs. Depending uponthe surface used, the bare surface may have the desired biophilic orbiophobic characteristics and, thus, the filling step may be omitted.When it is desired that cells adhere to a bare portion of some surfaces,the provision of serum facilitates this binding.

In one set of preferred embodiments of a patterned plate with a gridpattern, the islands are of a size and shape appropriate for bindingindividual cells and are separated one from another by a sufficientlylarge area of biophobic SAM so as to prevent cell-to-cell contact. Theislands of these plates are designed so as to be of a size less than orapproximately commensurate with the cells to be studied when the cellshave adhered. As is well known to one of ordinary skill in the art,cells in suspension will generally flatten upon adhering to a surface.Also, as in known in the art, cells vary in the degree to which theyflatten upon binding. White blood cells, for example, have a diameter ofapproximately 20 μm whereas Xenopus laevis oocytes may have a diameterof 1 mm. Relative to other cells, these cells do not flattensubstantially. On the other hand, most other cells, and particularlyepithelial cells, spread and flatten to a greater degree.

Endothelial cells for example, may have an area of approximately250-4,000 square μm, whereas hepatocytes may have an area ofapproximately 500-10,000 square μm. And, even within a given cell type,a wide range of sizes may be found. The appropriate size, therefore, isgenerally determined empirically. Beginning with an island size roughlycommensurate with the projected size of the cells when bound, it is wellwithin the ability of one of ordinary skill in the art to vary theisland size to determine a size appropriate to the intended use withoutundue experimentation. This is most easily accomplished by beginningwith a plate bearing islands of varying sizes, contacting the plate witha suspension of cells, and then determining which size or sizes ofislands appropriately bound cells. The size of the islands should bechosen such that it is not so large as to admit binding of more than onecell per island. In some circumstances, such as when it is desired toremove the cells by elution or for replica plating, a smaller size maybe chosen so that the cells have less contact area with the biophilicSAM and are more easily removed. The islands should not, however, be sosmall as to render cell adhesion unlikely.

In an alternative set of embodiments, the islands are chosen so as toadmit binding of a number of cells such that the cells may formcell-to-cell contacts. The size of the islands, however, is chosen so asto prevent formation of a large sheet of cells which would be subject tocell retraction or detachment, or the formation of spheroid and/ortrabecular structures of cells.

As is known to those of skill in the art, certain classes of cells,specialized epithelial cells in particular and especially anchoragedependent and polar cells, are affected by their binding to a substrateor their contacts with other cells. Thus, the viability, growth,proliferation, differentiation, orientation and spreading of certaincells have been shown to depend on the substrate to which the cells areadhered (D. Gospodarowicz, et al., Cancer Res., 3 8:4155-4171, 1978; J.Folkman and A. Moscona, Nature, 273:345-349, 1978; A. Ben Ze'ev. et al.,Cell, 21:365-372, 1980; D. E. Ingber, et al., In Vitro Cell Dev. Biol.,23:387-394, 1987; D. E. Ingber and J. Folkman, J. Cell Biol.,109:317-330, 1989). The growth and viability of anchorage dependentcells, for example, may be different when they are allowed to becomemore extended or flattened than when the cells are maintained in arounded form or in suspension. Similarly, for fibroblast growth factor(FGF) stimulated capillary endothelial cells, it has been demonstratedthat by altering the density of extracellular matrix (ECM) attachmentsites, cell shape is altered and the cells may be switched betweengrowth and differentiation modes in vitro (D. E. Ingber and J. Folkman,J. Cell Biol., 109:317-330, 1989). It has also been shown thatcell-to-cell contact or cell anchorage may affect cellular processessuch as post-translational modification of proteins (e.g. D. Kabat, etal., J. Cell Biol., 101:2274-2283, 1985).

Thus, the present invention also provides for patterned plates of cellswith altered or controlled viability, growth, proliferation,differentiation, protein processing, orientation, and/or spreadingcharacteristics.

Cells may be shaped using plates with islands of varying shapes whichare sufficiently large to bind only a single cell and which will causethe adhered cell to conform to that shape of the island. In this way, acell which normally flattens or extends may be forced to remain in arounded form. Alternatively, although an island may admit of bindingmore than one cell, the island may be shaped such that it is relativelynarrow in one dimension and forces the cells adhered to it to form asingle line of cells, each of which has been forced into an elongatedshape. The chosen area and shape of the island will depend upon theparticular cells and uses intended and, in light of the presentdisclosure, is within the ability and discretion of one of ordinaryskill in the art.

Alternatively, when cell-to-cell contact or anchorage in a moreflattened or extended form is desired in order to affect the viability,growth, proliferation, differentiation, protein processing, orientation,and/or spreading characteristics of cells, but large sheets of cells arenot desired because they are subject to detachment from the surface ofthe culture plate, islands admitting of more than one cell may be usedsuch that the cells establish the desired cell-to-cell contacts but donot form large sheets. In this embodiment, the islands are chosen to beof a sufficient size to adhere a desired number of cells but areseparated from other islands by biophobic regions which are sufficientlywide to prevent cells from bridging them. Such an embodiment isparticularly preferred for use in cultures in which the cells are keptor frequently washed with serum free media, as for example in abioreactor, and cell-to-cell contact is desired because of its effect onthe cells' viability, growth, proliferation, differentiation, proteinprocessing, orientation, and/or spreading characteristics. Bioreactorsinclude various devices for maintaining cultures such as perfusionsystems such as Cube System (Costar), T-Flasks such as the Falcon models(Bectin-Dickinson), roller bottle culture systems, and stirred tanks orspinner flasks with cells adhered to microcarriers or beads. The use ofthe plates of the present invention in bioreactors is particularlycontemplated.

In another embodiment of the invention using patterned plates with agrid pattern, the plates are employed in cytometry. For example, thenumbers or ratios of different types of cells in a sample may beefficiently assayed by contacting the suspension with one of the platesof the present invention, allowing a period of time for the cells tobind, washing away any excess solution or unbound cells if necessary,and then identifying and counting the different cell types at thespecific and predetermined locations of the biophilic islands. Becausethe size of the islands may be chosen such that no more than one cellmay bind on any given island, because the locations and geometricpattern of the islands may be predetermined, and because the cells willremain at fixed locations during the cell counting, the patterned platesof the present invention provide for much greater efficiency andaccuracy in cytometry.

In a particularly preferred embodiment in cytometry, the cells areidentified and counted by an automated detector unit. Because thelocations and geometric patterns of the islands are predetermined, thedetector can be designed or programmed to take measurements specificallyat those locations. The presence or absence of a cell on an island orthe nature of the cell may be detected by any of a variety of knownfluorescence or spectrophotometric assays based upon binding offluorescently labeled antibodies or other ligands, cell size ormorphology, or by the cells' spectrophotometric transmission, reflectionor absorption characteristics either with or without biologicalstaining. Standard light or electron microscopy can also be employed.The detector unit is positioned either above or below the plate. In thecase of fluorescence assays, a detector unit may be placed above theplate or, if the plate is translucent, below the plate. In the case oftransmission spectrophotometric assays, a translucent plate is used, asource of electromagnetic radiation is placed on one side of the plateand a detector unit on the other. Because of the small distances betweenindividual isolated cells permitted by the present invention, detectorsemploying fiber optics are particularly preferred. Such sources ofelectromagnetic radiation and such detectors for electromagnetictransmission, reflection or emission are known in the applicable art andare readily adaptable for use with the invention disclosed herein.

In one preferred embodiment of the assays described above, the detectorunit consists of a multiplicity of individual detectors in an arraycorresponding spatially to the islands of the plate such that the entiredetector unit may be positioned above or below the plate and theindividual detectors each measure electromagnetic radiation transmittedthrough or emitted or reflected from a particular island on the plate.In this embodiment, the number and type of a great many individual cellsmay be individually assessed simultaneously with minimal human labor orinvolvement. In another embodiment, the detector unit consists of asingle detector which may be sequentially positioned over each island tomeasure the electromagnetic emission or transmission of each cell,alternatively, or the plate may be moved to position each island underthe detector sequentially. Preferably, this sequential positioning isautomated and, in a most preferred embodiment, the detector isprogrammable such that it may be employed with plates of varyingdimensions and varied spacing between islands. The design of suchautomated detectors is well within the ability of one of ordinary skillin the applicable art.

When an automated detector unit is employed, a standard or control platemay also be provided. Such a plate would contain islands includingislands to which no cells are bound so that a reference would beprovided and the detector would recognize such islands. In addition,islands bearing cells of known types could be provided to act asreferences to allow the detector unit to classify the cells on a subjectplate. Furthermore, depending upon the biophilic SAM which is chosen,cells of different types may adhere to the plate with differingaffinities. Thus, depending upon the cells to be studied and thebiophilic SAM employed, a standard cytometric method may be employed ona sample first and then the plates and method of the present inventionmay be employed on the same or a substantially similar sample tocalibrate the system.

In addition, when only a subset of cells in a sample are of interest,for example, the white blood cells in a blood sample containing both redand white blood cells, a specifically biophilic SAM may be chosen thatwill selectively bind the cells of interest and, subsequent to binding,the extraneous cells may be washed away. Given a particular set orsubset of cells to be studied, the choice of a biophilic SAM specific tothose cells is within the ability of one of ordinary skill in the artand, given the disclosures herein, one of ordinary skill in the art isenabled to produce appropriate patterned biophilic SAMs specific forthose cells.

Merely by means of example, and without limiting the scope of thepresent invention, the following cytometric applications of the presentinvention are listed. The cytometry system provided by the presentinvention could be used in measuring the numbers and types of cells inblood, urine, cerebrospinal fluid, PAP smear, biopsy, ground water, seawater, riparian water, and reservoir water samples, and any otherapplication in which there is a desire to determine the presence, numberor relative frequency of one or more types of cells in a large sample ofcells.

In another aspect of the present invention, a method of assaying theeffects of various treatments and compounds on individual cells isprovided. In particular, the invention provides the capability to assaythe effects of various treatments or compounds on each of a great manyindividual cells plated at high density but separated from each otherand at fixed locations on the plate. In this embodiment of theinvention, many cells are applied in suspension to the plates of thepresent invention.

Once the suspension of cells has been applied to the plate, a period oftime is allowed to elapse in order to allow the cells to bind to theislands. Excess fluid including unbound cells may be washed away. Thecells may then be subjected to a treatment or exposed to a compound insitu on the plate or, in some situations, the cells may be pre-treatedbefore being introduced to the plate for binding. The effects of thetreatment or compound on each cell may then be individually assayed in amanner appropriate to the cell type and the treatment or compound beingstudied. For example, the effects of treatments or compounds potentiallycapable of affecting cell morphology may be assayed by standard light orelectron microscopy. Alternatively, the effects of treatments orcompounds potentially affecting the expression of cell surface proteinsmay be assayed by exposing the cells to either fluorescently labeledligands of the proteins or antibodies to the proteins and then measuringthe fluorescent emissions associated with each cell on the plate. Asanother example, the effects of treatments or compounds whichpotentially alter the pH or levels of various ions within cells may beassayed using various dyes which change in color at determined pH valuesor in the presence of particular ions. The use of such dyes is wellknown in the art. For cells which have been transformed or transfectedwith a genetic marker, such as the B-galactosidase, alkalinephosphatase, or luciferase genes, the effects of treatments or compoundsmay be assessed by assays for expression of that marker and, inparticular, the marker may be chosen so as to causespectrophotometrically assayable changes associated with its expression.

In particularly preferred embodiments, the assay is spectrophotometricand automated. In these embodiments, the treatment or compoundpotentially causes a change in the spectrophotometric emissions,reflection or absorption of the cells. A detector unit, as describedabove, may be employed. Because of the small distances betweenindividual isolated cells permitted by the present invention, detectorsemploying fiber optics are particularly preferred. Such sources ofelectromagnetic radiation and such detectors for electromagnetictransmission, reflection or emission are known in the applicable art andare readily adaptable for use with the invention disclosed herein.

In one preferred embodiment of the assays described above, the detectorunit consists of a multiplicity of individual detectors in an arraycorresponding spatially to the islands of the plate, as described above.In this embodiment, the effect of a treatment or compound on a greatmany individual cells may be individually assessed simultaneously withminimal human labor or involvement.

In particularly preferred embodiments, a suspension of cells is appliedto one of the plates of the present invention in which the biophilic SAMis chosen so as to selectively or preferentially bind a certain type ortypes of cells. The cells are subjected to a treatment or exposed to acompound which will potentially cause a change in the electromagneticemission, reflection or transmission characteristics of the cells and anautomated detector unit records the emission, reflection or transmissioncharacteristics of each cell individually by assaying electromagneticemission, reflection or transmission at points corresponding to eachisland on the plate.

When an automated detector unit is used, a plate which has not beenexposed to any cells may be used as a control before testing theexperimental plate to provide reference values to exclude from theresults islands on the experimental plates which have been exposed tocells but which have not bound cells.

In another particularly preferred embodiment, plates upon which cellshave been allowed to bind are assayed prior to any potentially effectivetreatment or compound and then treated or exposed. As the cells maintaintheir individual positions on the plates, a second assay may beperformed to detect changes in the assay results on a cell-by-cell basisafter treatment or exposure. Such a two-step assay is particularlyappropriate for treatments or compounds which potentially cause celltoxicity or disrupt binding.

The above described embodiments, employing the plates of the presentinvention which allow for plating individual cells at high density butwith little or no overlap or contact of cells, can be employed for highthrough-put tests of potentially useful treatments including radiationand pharmacological or toxicological compounds. In particular, thepresent invention provides assays which allow assays both as toqualitative and quantitative changes in individual cells andquantitative assays as to percentages of cells affected by any giventreatment or compound.

In a different embodiment, the present invention provides means foridentifying individual cells which have been successfully transformed ortransfected with recombinant DNA technology. A culture of cells exposedto transforming or transfecting vectors, including plasmids, phasmids,cosmids, retroviruses and various homologous recombination orintegration elements, may be plated on the plates of the presentinvention to separate the cells and cause them to bind individually atthe locations of the islands on the plate. Individual cells which havebeen transformed or transfected may then be identified by the methodsdescribed above or other methods well known to those of ordinary skillin the art. Particularly simple, given the disclosures herein, is theidentification of individual cells transformed or transfected with avector including a marker locus which causes a spectrophotometricallydetectable change in a cell's function, metabolism, gene expression ormorphology. Marker loci may also be included which cause cells toexhibit a sensitivity or resistance to a particular treatment orcompound.

Cells transformed or transfected by such vectors may be first selectedon the basis of the appropriate sensitivity or resistance and thenplated as individual cells and further selected or characterized by themethods and employing the plates described herein. In particular,selection may be employed prior to plating on the plates of the presentinvention to isolate transformed or transfected cells and then the cellsmay be assayed in situ using the presently disclosed materials andmethods to identify and isolate cells with, for example, particularlyhigh or low expression of the characteristic to which the transformationor transfection was directed.

In a particularly preferred embodiment, an automated detector unit, asdescribed above, is employed.

In a different embodiment, the present invention provides materials andmethods for retrieving individual cells which are bound to the plates ofthe present invention. That is, the present invention provides formaterials and methods for isolating and manipulating particularindividual cells which are present on a plate containing a greatmultiplicity of cells separated one from another by only a few microns.

In one embodiment, cells are plated to a primary plate with each islandof the plate capable of binding a single cell. A secondary plate,bearing an island with an appropriate biophilic SAM only at a positioncorresponding spatially to the position of the island bearing thedesired cell may then be contacted with the cell on the primary plate.The desired cell may then adhere to the secondary plate such that, whenthe secondary plate is drawn away from the primary plate, the desiredcell and only the desired cell adheres to the secondary plate. Thus, theindividual desired cell is retrieved for purposes which may includegrowth for a DNA or cDNA library, growth into a tissue culture, or inthe case of transformed or transfected oocytes, uterine implantation andgrowth into a transgenic organism. The secondary plates of the presentinvention may be custom made to retrieve a particular cell by means ofthe stamp production methods disclosed herein, or may be pre-made. If,for example, plates of a particular dimension with islands positioned atstandard points are employed on a regular basis, secondary plates forretrieval of cells bound at particular islands may be prepared inadvance. Thus, for a plate with 100 islands in a 10×10 array, 100secondary plates may be prepared in advance for retrieval of anindividual cell at any one of the 100 possible island positions. Moreefficiently, 25 secondary plates may be prepared in advance to contactwith any of the four 5×5 quadrants of a 10×10 primary plate. Even moreefficiently, if the pattern is symmetrical, a lesser number of secondaryplates capable of rotation may be produced. In addition, secondaryplates could be produced which would retrieve more than one cell byconstructing a secondary plate with biophilic SAM islands correspondingspatially to more than one island on the primary plate. Further, toenhance transfer of the desired cell or cells from the primary plate tothe secondary plate, it may be desirable to have larger islands ofbiophilic SAM on the secondary plate or to use a biophilic SAM on thesecondary plate with a higher binding affinity for the desired cell orcells than the biophilic SAM on the primary plate.

In another embodiment of a cell retrieval system, an automated systemprovides for the contacting of a secondary plate with a specified islandof a primary plate such that a desired cell is transferred from theprimary to the secondary plate. Thus, a desired cell is identified bythe coordinates of the position of the island to which it is bound and asecondary plate, consisting of a single island is positioned over thecoordinates of the desired cell on the primary plate and the secondaryplate is contacted with the desired cell to allow transfer of thedesired cell from the primary to the secondary plate. As above, the sizeof the island or the nature of the biophilic SAM on the secondary platemay be chosen so as to enhance the efficiency of transfer of the desiredcell from the primary to the secondary plate. In a particularlypreferred embodiment, the detection of the desired cells is by means ofan automated detector unit and the coordinates of the desired cells aretransmitted from the detector unit to an automated retrieval unit suchthat human labor and involvement are minimized.

Given the disclosures of the present invention for isolating individualcells on islands at predetermined positions on one of the disclosedplates, the design and production of a cell retrieval unit is within theability of one of ordinary skill in the applicable art. Absent thepresent disclosure, retrieval of a particular individual cell fromamongst a high density plate of a great many cells would be an arduousand difficult task. The binding of individual cells to particularlydefined positions on the plates of the present invention, however,provides for a method of such retrieval. Such a cell retrieval systemmay be employed, for example, to retrieve transformed or transfectedcells, potentially cancerous cells in a PAP smear or biopsy, orfertilized eggs adhered to the patterned plates of the presentinvention.

In another aspect of the present invention, patterned plates and amethod are provided for immobilizing cells for microinjection. As isknown in the art, microinjection of, for example, dyes, proteins, andDNA or RNA sequences, is made more difficult when the cells to bemicroinjected are not immobilized on a substrate and/or localized atspecific and predetermined positions. By providing the patterned platesand methods disclosed herein, the present invention greatly simplifiesthe microinjection process. Thus, in light of the present disclosure,patterned plates with biophilic islands which can bind a given type ortypes of cells can be produced and the type or types of cells can bebound individually to specific and predetermined locations on theplates. Cell types which may be sought to be bound include bacterialcells such as Escherichia and Pseudomonas species; mammalian cells suchas chinese hamster ovary (CHO), baby hamster kidney (BHK), COS, humanfibroblast, hematopoietic stem cells, and hybridoma cell lines; yeast;fungi; and cell lines useful for expression systems such as yeast orXenopus laevis oocytes. The listing above is by no means intended to beexhaustive but is merely exemplary of the sorts of cells which may beimmobilized to specific and predetermined positions for microinjection.Subsequent to microinjection, the cells may be assayed for functionalexpression or transformation on the plates of the present invention withthe detectors described herein and, if desired, individually retrievedwith the retrieval system disclosed herein.

In another aspect of the present invention, materials and methods areprovided which allow for the immobilization of oocytes at specific andpredetermined positions for in vitro fertilization techniques. That is,the patterned plates of the present invention allow for immobilizationof oocytes, including human oocytes, at specific and predeterminedpositions. These immobilized oocytes may then be contacted in situ onthe plates with a solution including sperm cells potentially capable offertilizing the oocytes. The fertilized oocytes, or zygotes, may then beconveniently identified because of their fixed positions on the platesof the present invention and individually retrieved for implantation orstorage by standard methods or the methods disclosed herein. As will beobvious to one of ordinary skill in the art, the biophilic SAM forimmobilizing the oocytes/zygotes can be chosen from a wide array ofpotential SAMs ranging from generally biophilic or hydrophobic SAMs toSAMs including moieties, including antibodies, which specifically bindthe oocytes/zygotes involved in the in vitro fertilization process.Subsequent to exposure to the sperm solution, the cells may be assayedfor successful fertilization on the plates of the present invention withthe detectors described herein and, if desired, individually retrievedwith the retrieval system disclosed herein.

In another aspect of the present invention, patterned plates areprovided which may be used to bind or adsorb proteins in specific andpredetermined patterns.

As is known to those of ordinary skill in the art, phenomena associatedwith the adsorption of proteins to solid synthetic materials areimportant in many areas of biotechnology including, for example,production, storage and delivery of pharmaceutical proteins,purification of proteins by chromatography, design of biosensors andprosthetic devices, and production of supports for attached tissueculture (see, for example, ACS Symposium Series 343, T. A. Horbett andJ. L. Brash, Eds., Am. Chem. Soc., Washington, D.C., 1987; J. D.Andrade, Surface and Interfacial Aspects of Biomedical Polymers: ProteinAdsorption, Plenum Press, NY, 1985; Materials Research SocietyProceedings 252, L. G. Cima and E. Ron, Eds., Mat. Res. Soc.,Pittsburgh, Pa., 1992). A number of researchers have demonstrated theformation of patterns of proteins (see, for example, A. S. Lea, et al.,Langmuir 8:68-73, 1992). But these have typically relied onphotolithography to create the patterns (see, for example, S. K. Bhatia,et al., J. Am. Chem. Soc., 114:4432-4433, 1992; S. K. Bhatia, et al.,Anal. Biochem., 208:197-205, 1993). The patterned plates of the presentinvention provide for relatively inexpensive and efficient patterning ofproteins with features of the pattern as small as 0.2-1 μm which areuseful in these applications.

In this embodiment, a plate is created with patterned SAMs according tothe methods disclosed above. Depending upon the desired application, thepattern may include islands or parallel rows of SAMs with differentproperties. One SAM may be biophilic and the other may be biophobic asapplied or they may be chemically modified as described above so as tobecome biophilic or biophobic subsequent to SAM formation. In analternative embodiment, as described above, two SAMs may be patternedonto a plate, one of which is biophilic and one of which is biophobicand, subsequent to binding a protein or proteins to the biophilic SAM,the biophobic SAM may be chemically modified so as to become biophobic.In this way, a second protein or group of proteins may be bound to thepreviously biophobic but now biophilic SAM to create a pattern of two ormore protein groups. Similarly, patterns of more than two SAMs may beused to create more complicated patterns of proteins in accordance withthe present invention.

For protein adsorption, particularly preferred biophilic SAMs are thosecomprised of ω-functionalized alkanethiols (HS(CH₂)_(n)R) where the Rgroup is a non-polar (e.g., R═CH₃) or ionic (e.g., R═CO₂ ⁻, PO₃H⁻, or2-imidazolo) group. Particularly preferred biophobic SAMs areω-functionalized alkanethiols (HS(CH₂)_(n)R) in which the R group is apolar but non-ionic group such as oligo (ethylene glycol)-terminatedthiols (e.g., R═(OCH₂CH₂)₆OH).

Patterned proteins which have been patterned according to the materialsand methods of the present invention may be transferred from thepatterned plates of the present invention by contacting the plates withother biophilic or bioadhesive substrates, well known in the art,without disrupting the patterns and then be used in a variety ofapplications. Thus, for example, the surfaces of prosthetic or implanteddevices or tissue culture plates can be patterned with the patternedproteins produced by the present invention. Alternatively, such devicesmay be directly stamped with the SAM patterns of the present inventionand the proteins patterned directly upon them.

In a different embodiment of the present invention, the patterned platesprovided herein may be used to produce plates with cells growing indesired patterns and to control the growth, proliferation,differentiation, orientation and/or spreading of certain classes ofcells.

As in the previously described embodiments of the present invention,depending upon the intended use, an enormous variety of patterns may beproduced and a multiplicity of stamps and/or a multiplicity of SAMs maybe employed to create patterns of one or more types of cells. As before,the SAMs may be generally or specifically biophilic or biophobic asapplied or may be subsequently modified to become generally orspecifically biophilic or biophobic after SAM formation by chemicalmodification of functional groups at the free ends of the SAM-formingcompounds. In particular, when several SAMs are present in a pattern butonly one is biophilic, a first type of cell may be adhered to thebiophilic SAM and then one of the remaining biophobic SAMs may bechemically modified in situ so as to become biophilic. A second celltype may then be adhered to the newly biophilic SAM and this process canbe repeated to create a complex pattern of different cell types.

In another embodiment, a plate with patterned proteins may be preparedas described above and cells may then be allowed to adhere to thepatterned proteins to form a plate of patterned cells. In particularlypreferred embodiments, the proteins are extracellular matrix proteinssuch as collagen, fibronectin or laminin; or are specific cell receptorssuch as integrins. In these embodiments, then, the patterned proteinmediates the cell adhesion to form patterned cells. Alternatively, apatterned plate of biophilic and biophobic SAMs may be created and awide variety of non-protein compounds may first be adhered to thepattern to mediate cell binding. Such compounds include but are notlimited to sialic acid, lectins, polygalactose and other carbohydrates.

The patterned plates of the present invention may be used to createpatterns of cells in which cells are isolated on islands to prevent cellto cell contact, in which different types of cells are specificallybrought into contact or in which cells of one or more types are broughtinto a pattern which corresponds to the pattern or architecture found innatural tissue.

Such plates of patterned cells have a wide variety of applications whichwill be apparent to one of ordinary skill in the art and all suchapplications are intended to fall within the scope of this invention.Particularly preferred applications include but are not limited to usein bioreactors for the production of proteins or antibodies, especiallyby recombinant cells; use in tissue culture; use for the creation ofartificial tissues for grafting or implantation; use in artificialorgans such as artificial liver devices for providing liver function incases of liver failure; and use for generating artificial tissues toadhere to the surfaces of prosthetic or implantable devices to preventconnective tissue encapsulation.

In another embodiment particular different types of cells may be broughttogether on the same plate. For example, in the toxicological assaysdescribed above, it may be desired to plate a percentage of hepatocyteson the plate to convert potentially procarcinogenic compounds intocarcinogenic compounds and to assay the effects on other nonhepatocytecells on the same plate.

In another embodiment of the present invention, specific cell to cellcontacts can be patterned by using a plate with island-like biophilicregions which are connected by biophilic bridge-like regions but areotherwise surrounded by biophobic material. Such a “bridged island”plate would have application, for example, in creating patterned neuralcells with neural processes making contacts only along specific andpredetermined bridge regions.

In a different embodiment of the invention, the present invention mayprovide one or more microcultures of determinable volume, and specificand predetermined positions on a plate. For example, if the free ends ofa first SAM-forming compound are hydrophilic and the free ends of asecond SAM-forming compound are hydrophobic, and these compounds areused to form a patterned plate in which there are small islands of thehydrophilic SAM, a plurality of droplets of aqueous fluid may be placedon these islands. With knowledge of the surface area of the islands, andthe surface tension of the aqueous fluid and hydrophobicity andhydrophilicity of the islands and surrounding SAM (measured, forexample, by contact angle), the volume of the droplets may bedetermined. Thus, if the volume of the droplet microcultures is knownand/or can be controlled, and the concentration of compounds within themicrocultures can be controlled, then individual cells, includingbacteria, yeast, and mammalian, as well as spores and othermicroorganisms can be cultured in these microcultures. Such amicroculture may be used in the same means and with the same assays anddetector and retrieval units as described above. In addition, however,such microcultures are particularly useful for assays for compounds thatare secreted from cells. Because the cells are isolated in their ownmicrocultures, such compounds do not diffuse away or mix with compoundssecreted by other cells in nearby but isolated microcultures.

The methods and materials, function and advantage of these and otherembodiments of the present invention will be more fully understood fromthe examples below. The following examples are intended to illustratethe benefits of the present invention, but do not exemplify the fullscope of the invention.

EXAMPLE 1 Preparing a Mold and Stamp

A mold according to the present invention was fabricated. A templateconsisting of an exposed and developed photoresist pattern on silicon isprepared (This type of fabrication is described in any conventionalphotolithography text, such as Introduction to MicroelectronicFabrication, by Richard C. Jaeger, Gerold W. Neudeck and Robert F.Pierret, eds., Addison-Wesley, 1989). Templates such as electronmicroscopy grids or other corrugated materials may also be used. Thetemplate is placed in a container such as a petri dish. A 10:1 (w:w orv:v) mixture of PDMS—Sylgard Silicone Elastomer 184 and Sylgard CuringAgent 184 (Dow Coming Corp., Midland, Mich.) was poured into the petridish. It was not necessary to put the mixture of PDMS-elastomer andcuring agent under vacuum to remove dissolved dioxygen. The PDMS curedat room temperature in the laboratory ambient for 30 to 60 min.

This cure was followed by additional curing at 65° C. for approximatelyone hour or until the polymer was rigid. After cooling to roomtemperature, the PDMS-stamp was carefully peeled from the template.

EXAMPLE 2 Preparation of Plates with Metallic Surfaces

Gold films (˜2000 A thick) were prepared by electron-beam evaporation ofgold (Materials Research Corp., Orangeburg, N.Y.; 99.999%) ontosingle-crystal silicon (100) test wafers (Silicon Sense, Nashua, N. H.;100 mm dia., ˜500 μm thick) that had been precoated with a film oftitanium (Johnson Mathey, 99.99%; ˜50 A thick) that acted as an adhesionpromoter between the silicon oxide and the gold. The silicon waferscoated with gold were fractured into square plates (˜2 cm×2 cm) and usedin the formation of the various types of patterned SAM plates.

EXAMPLE 3 Preparing a Patterned Plate with a Grid Pattern

A stamp fabricated in accordance with Example 1 was fabricated. Thestamp was fabricated so as to have a linear indentation patterncontiguous with a liner stamping surface pattern. That is, the stamp hadan array of indentational lines separating stamping surface lines. Thesurface was coated with hexadecanethiol in ethanol using a cotton swab.The stamp was applied to a smooth gold surface, and removed. Theresultant pattern included parallel SAM lines of 2 microns in width.After removal of the stamp, the stamping surface was re-coated androtated approximately 45° to a second orientation, and re-applied to thesurface. A grid pattern resulted.

EXAMPLE 4 Patterned Proteins

Several patterned SAM plates were prepared by first forming patterns ofHS(CH₂)₁₅CH₃ on gold using a rubber stamp made of polydimethylsiloxane.The remaining, underivatized surface of the gold was derivatized with asecond SAM by placing the patterned plate in a second solutioncontaining HS(CH₂)₁₁EG₆OH (10 mM in ethanol) for 10 s. The sample wasrinsed with heptane and with ethanol and dried under a stream of drynitrogen.

Buffer solutions for protein adsorption were prepared from potassiumdihydrogen phosphate (0.01 M) and titrated to pH 7.5 with sodiumhydroxide (0.1 M). Briefly, the protocol for effecting adsorption ofproteins involved the immersion of plates with patterned SAMs insolutions of the protein of interest in phosphate buffer at roomtemperature. After the desired time of immersion, the plate was removedfrom the solution and rinsed with distilled, deionized water and driedunder a stream of nitrogen. To pattern an adsorbed protein, it wasdesirable to use a modification of the protocol of Horbett, (Horbett, T.A. in Techniques of Biocompatibility Testing Vol. II, pp. 183-214,Williams, D. F., Ed., CRC Press Inc. Boca Raton, Fla., 1986; Rapoza, R.J. and Horbett, T. A. J. Coll. Interface Sci., 136:480-493, 1990), andfirst to place the patterned SAM plate in buffer and then to addconcentrated protein solution so that the final, total concentration ofprotein in the solution in contact with the patterned SAM plate was thatdesired for adsorption (e.g., 1 mg/mL). This procedure eliminated theexposure of the SAM to the interface of the solution of protein and air.After the appropriate adsorption time, the solution of protein wasdisplaced with at least five equivalent volumes of distilled deionizedwater. The samples were further rinsed directly with distilled deionizedwater and dried under a stream of nitrogen.

EXAMPLE 5 Patterned Plating of Primary Rat Hepatocytes

Mold patterns were developed using a computer program commonly used forintegrated circuit designs. The pattern was created on a glass platecoated with photosensitive emulsion using a pattern generator (Gyrex1005A). The final mask was made by contact printing the emulsion plateonto a chromium-coated quartz plate. Silicon wafers were cleaned inpiranha solution, rinsed with distilled water and dehydrated under N₂ at200° C. for 8 hrs. One layer (1-2 μm) of negative photoresist (UnionCarbide) was spin-coated on the silicon wafers. The coated wafers werebaked at 90° C. for 30 min, placed in a mask aligner (Karl Zuss, Munich,Germany), and exposed to ultraviolet light through the chromium maskcontaining the desired pattern. The exposed wafers were hard baked at120° C. for 90 s and developed to produce the desired photoresisttemplate for the rubber stamp. A 10:1 mixture (v/v or w/w) of SiliconeElastomer-184 and Silicone Elastomer Curing Agent-184 (Dow ComingCorporation) was poured onto this photoresist template, allowed to cureat 60° C. for 45-90 min, and peeled away from the template. Theresulting PDMS stamp was inked by brushing it with a cotton swab thathad been moistened with a 1 mM solution of a hexadecanethiol(HS(CH₂)₁₅CH₃) in ethanol. The inked stamp was then placed in contactwith a plate with a gold surface (2000 Angstroms Au on a titanium-primedsilicon wafer) to form the SAMs of hexadecanethiolate. Upon removal ofthe stamp, the plate was washed for 5 s in a solution of an alkanethiolterminated with ethylene glycol oligomer (HS(CH₂)₁₁(OCH₂CH₂)₆OH, 1 mM inethanol). The plate was finally washed with ethanol and dried in astream of dry nitrogen. Patterned plates were coated with 1 μg/cm² ofmouse laminin using a carbonate buffer adsorption method. The lamininadhered to the hexadecanethiol islands but not to the hydrophilicPEG-terminated SAM. Primary rat hepatocytes were isolated and culturedin defined medium on the laminin-coated islands as described in Mooneyet al., J. Cell Physiol., 151:497 (1992).

EXAMPLE 6 Controlling Cell Shape and Function

The ability of the techniques of the present invention to controldistribution and shape was explored by plating primary rat hepatocytesin hormonally-defined medium (Mooney et al, Id.) on laminin-coatedsubstrata that were stamped with square and rectangular islands havingmicron dimensions (5×5 microns to 100×100 microns). Cells attachedpreferentially to the adhesive, laminin-coated islands and wereprevented from extending onto surrounding non-adhesive regions.Selective attachment of cells to these adhesive islands resulted indevelopment of regular arrays of individual adherent cells that werealigned single file in both horizontal and vertical columns, extendingover large areas (mm²) of the culture surface. The adhesive islandslimited cell spreading and largely prevented cell-cell contactformation, although a few cells (less than 10%) were found to bridgebetween adjacent islands. In most cases, cells conformed to the shape ofthe underlying island. Actual projected cell areas, determined bycomputerized image analysis, were consistently within 20% of the islandarea. In many cells, the abrupt prevention of cell spreading at thelateral borders of each island resulted in formation of correspondingsquare and rectangular cells with “corners” that approximated 90degrees. In contrast, cells plated at the same density on unpatterned,laminin-coated gold substrata spread extensively, formed numerouscell-cell contacts, and exhibited normally pleiomorphic shapes. Thus,the shape and spatial distribution of cultured cells could be simplycontrolled using our “rubber stamp” method to engineer adhesive islandswith desired design characteristics and shapes.

Since the extent to which a cell spreads influences its growth andfunction, maintaining large populations of cells in specific shapes onpatterned substrata can be an effective way of controlling both thebehavior of individual cells and the performance of the entire culture.By fabricating adhesive islands of varying size on a single substratum,we were able to maintain cells at defined degrees of extension forextended periods of time, independent of the presence of growth factorsor the composition mechanics or molecular density of the extracellularmatrix. Quantitation of DNA synthesis in hepatocytes cultured on islandsof different size in defined medium (Mooney et al., Id.) containingsaturating amounts of soluble growth factors (EGF, insulin, anddexamethasone) revealed significant differences in the number of cellsthat exhibited nuclear labelling. DNA synthesis was highest (60% nuclearlabeling index) in cells on unpatterned surfaces where cells couldspread without restriction. Decreasing the size of the adhesive islandsresulted in a progressive reduction in growth such that less than 3% ofthe cells adherent to the smallest islands (less than 1600 μm²) enteredS phase. This result was not due to changes in cell viability; similarpercentages of cells incorporated the vital dye, calcein AM, on allsubstrata.

We assessed the differentiated function of hepatocytes cultured onsubstrata coated with uniformly sized islands by assaying the culturesupernatant for secretion of albumin, a liver-specific product.Hepatocytes cultured on unpatterned substrata rapidly lost the abilityto secrete high levels of albumin. In contrast, hepatocytes maintainednear normal levels of albumin secretion for at least 3 days whencultured on the smallest adhesive islands (40×40 μm) that fullyrestricted cell extension. In general, albumin secretion rates decreasedas the size of the adhesive island was increased and growth waspromoted. These data are consistent with studies, that show that cells,such as hepatocytes, can be switched between growth and differentiationin the presence of soluble mitogens by modulating cell shape. In paststudies, shape was controlled by varying ECM molecular coating densitieson otherwise non-adhesive dishes and thereby, altering cell-ECM contactformation. The present results demonstrate that it is the degree towhich the cell extends and not the density at which the ECM ligand ispresented that indicates whether a cell will grow or differentiate. Useof this type of approach, which relies upon chemically-defined,patterned surfaces and serum-free culture conditions, facilitatesfurther analysis of the basic mechanism of coupling between cell shapeand function and of other fundamental biological processes that involvechanges in cell form or cell-cell contact formation.

1. A device for adhering at least one cell in a specific andpredetermined pattern comprising: a surface; and a plurality of islandsin a specific and predetermined pattern over the surface that adherecells to the islands, the islands isolated from each other by a regioncontiguous with the islands and to which the cells do not adhere, andwherein the islands or the region in which the cells do not adhere orboth comprise a self-assembled monolayer.
 2. The device of claim 1,wherein the region in which the cells do not adhere or the islandscomprise more than one self-assembled monolayer.
 3. The device of claim1, wherein the surface is defined by a plate.
 4. The device of claim 3,wherein the plate is transparent to electromagnetic radiation.
 5. Thedevice of claim 1, wherein at least one of the plurality of islandscomprises a self-assembled monolayer.
 6. The device of claim 1, whereinthe region in which the cells do not adhere comprises a self-assembledmonolayer.
 7. The device of claim 1, wherein the islands are located ina plurality of predetermined positions on the surface.
 8. The device ofclaim 1, wherein at least one of the plurality of islands binds only aselected cell type.
 9. The device of claim 1, wherein the islands areable to adhere one cell type but are not able to substantially adhere asecond cell type different from the first cell type.
 10. The device ofclaim 1, wherein the plurality of islands includes a first island ableto adhere a first population of cells and a second island able to adherea second population of cells different from the first population ofcells.
 11. The device of claim 1, wherein at least one of the pluralityof islands has a predetermined shape that is able to influence the shapeof a cell adhered thereto.
 12. The device of claim 1, wherein theislands are sufficiently isolated to prevent cells adhered to theislands from contacting each other except via formation of cellularbridges above and free of adhesive contact with the region in which thecells do not adhere.
 13. The device of claim 1, wherein at least one ofthe plurality of islands has a size chosen such that only an individualcell is able to adhere thereto.
 14. The device of claim 1, wherein atleast one of the plurality of islands has a size sufficient to allow aplurality of cells to adhere thereto.
 15. The device of claim 1, whereinat least one of the plurality of islands is between 1 and 2,500 squaremicrons.
 16. The device of claim 1, wherein at least one of theplurality of islands is between 1 and 500 square microns.
 17. The deviceof claim 1, wherein at least one of the plurality of islands is between1 and 100 square microns.
 18. The device of claim 1, wherein at leastone of the plurality of islands has a lateral dimension of between 0.2and 10 microns.
 19. The device of claim 1, wherein at least one of theplurality of islands is elongated.